Reactive probe chip, composite substrate and method for fabrication of the same

ABSTRACT

A reaction probe chip which is prepared by loading a reactive probe on fine pieces of carrier such as particles, tile-like plates and then arraying and immobilizing the reactive probe-loaded carrier on a base material. The carrier fine pieces such as particles, tile-like plates and the like are porous or have a reactive surface, and the base material is preferably a thin inorganic plate or a thin organic plate is disclosed. The inorganic base material is preferably a glass slide or silicon wafer, and the organic base material is preferably a polyester film or polyethylene film. In case the porous carrier pieces are used, the reactivity of the inner surfaces of the porous carrier pores should be maintained during array or immobilization process of the reactive probe-loaded carrier. A composite substrate characterized in that on at least a section of the surface thereof, a plurality of porous regions are orderly arranged as compartments by non-porous regions, or a plurality of non-porous regions are orderly arranged as compartments by porous regions is also disclosed. The porous solid is preferably porous glass or porous ceramic, the porous glass is preferably split-phase porous glass, and the surface is preferably flattened by a process such as polishing.

BACKGROUND OF THE INVENTION

The present invention relates to a reactive probe chip capable ofrecognizing a plurality of functional molecules, to be used for genediagnosis, physiological function diagnosis and the like, and to amethod for its fabrication.

Also, the present invention relates to a composite substrate wherein onat least a section of the surface thereof, a plurality of porous regionsare arrayed as compartments encompassed by non-porous regions, or aplurality of non-porous regions are arrayed as compartments encompassedby porous regions, and to a method for its fabrication.

Detection of polymorphisms due to gene mutations, and particularlysingle base (codon) mutations, is not only effective for diagnosis ofcancer and other diseases resulting from mutations, but is alsonecessary for indication of drug responsiveness and side-effects, andcan be helpful for the analysis of the causative genes of multiplefactor diseases, and for predictive medicine. The use of “DNA chips” fordetection is known to be effective. The hitherto utilized “Gene Chip” byAffymetrix, which is a DNA chip containing immobilized short DNA chains,usually comprises over 10,000 oligo DNA fragments (DNA probes) mountedon an approximately 1 cm square silicon or glass plate using aphotolithographic technique.

When a fluorescently-labeled DNA sample to be examined is allowed toflow over the DNA chip, the DNA fragments having sequences complementaryto the probes of the DNA chip bind to the probes to perform detection ofonly those sections by their fluorescence, and thereby detecting andquantifying the specific sequences of DNA fragments in the DNA sample.It has already been demonstrated that this method can detect cancer genemutations and gene polymorphisms.

Microarrays with cDNA immobilized on slide glass may also be used.

On the other hand, porous solids have been widely used in the prior artas carriers for catalysts, enzymes, microorganisms and the like, and areutilized as sites for various reactions. They are also used asfunctional materials for adsorption and separation, or as materialsproviding low heat or electrical conductivity. Porous glass or porousceramics are used as porous solids, and methods of controlling the poresizes or imparting functional groups have provided properties suitablefor specific uses.

There were three principal problems in the prior art. DNA chipsemploying photolithography require at minimum four photomasks for eachstep of synthesis, and the photolithography, coupling and washing mustbe repeated four times. Since this is repeated the required timesdepending on the chain length, the cost is high (problem 1). Also, it isnecessary to change each photomask in order to change the pattern, forwhich reason, DNA chips with various designs could not be flexiblyfabricated (problem 2).

That is, although the types of DNA probes to be immobilized on the DNAchip are previously decided and although it is easy to fabricate andsupply such DNA chips at locations where the necessary equipment isavailable, different photomasks must be prepared for synthesis of eachbase in the probe. Therefore, the reaction process has may steps and itis difficult to fabricate in a flexible manner, DNA chips containing DNAprobes for different purposes. High costs are also incurred. When thenumber of DNA probes required is small, the degree of integration of theDNA probes on the chip need not be so high. Rather, it is sometimesdesirable for the chip to have the desired DNA probes immobilized in amore convenient manner. The DNA chip must also be provided at low costand with high stability if it is clinically used to detect DNApolymorphisms of individuals.

Alternatively DNA Microarray chips prepared by spotting a solution ofsynthesized oligonucleotide at high density, is proposed. In thisprocess, modifying groups are introduced after synthesis of theoligonucleotides and then the modified oligonucleotides are cut out andreleased from the carrier and purified. Further the purifiedoligonucleotides are reacted with the functional groups previouslyintroduced onto the substrate glass. Thus, the process is very complexand the cost is therefore high (problem 3) as of the DNA chips producedby photolithography.

With respect to substrates for probe chips, porous solids have a widevariety of applications, for example, carrier of probe chips such as DNAchips, and the like, but the use of porous solids having continuousporous regions throughout the entire surface has been limited in thatonly one function can be loaded on such a single and uniform substrate.

However, in recent advancements in scientific techniques, downsizeddevices is in ever greater demand and it has been desired to develop acomposite substrate having unique utilities not found in the prior art,and a substrate having different reaction sites with a plurality offunctions on the single substrate whereby the local areas of thesubstrate can be thermally or electrically insulated each other.

In light of the problems in the prior art, it is an object of thepresent invention to provide a composite substrate as a single substrateallowing a plurality of different functions to be loaded, as well as amethod for its fabrication.

SUMMARY OF THE INVENTION

The present invention solves the aforementioned problems with respect toreactive probe chips and to composite substrates by way of the followingmeans.

According to one aspect of the invention, porous carrier particles areused as a carrier fine pieces to be loaded with reactive substances orreactive probes.

(1) A reactive detecting chip characterized in that fine porous carrierparticles having reactive substances, which have ability to bonddifferent detection targets and which are loaded on the inner surfacesof the porous particle pores, serve as a whole porous carrier particleprobes. They are arrayed and bound or immobilized on at least one of aplurality of microcompartments provided on a base material, whilemaintaining the reactivity of the inner surfaces of the porous carrierparticle pores.

(2) A reactive probe chip according to (1) above, wherein the porouscarrier particles loaded with the reactive probes are of a materialhaving a bonding surface, such as porous glass, silica gel orion-exchange resin.

(3) A reactive probe chip according to (1) or (2) above, wherein thepore size of the porous carrier particles ranges from 10 nm to 1 μm, andthe particle size is from 1 μm to 100 μm.

(4) A reactive probe chip according to any one of (1) to (3) above,wherein the base material immobilizing the porous carrier particlesloaded with the reactive is an inorganic base material or organic basematerial.

(5) A reactive probe chip according to any one of (1) to (4) above,wherein the reactive probes to be loaded in the porous carrier particlesare DNA, RNA or PNA (peptide nucleic acid) or fragments thereof,oligonucleotides of any desired base sequence, antigens, antibodies orepitopes, or enzymes, proteins or their partial polypeptide chainshaving the target functions.

(6) A method for fabrication of a loaded porous carrier particleswherein a solid phase method is used to synthesize oligonucleotideshaving desired base sequences or proteins having desired structures onporous carrier particles, and they are used as they are.

(7) A method for fabrication of a loaded porous carrier particles, whichcomprises binding DNA, RNA or PNA (peptide nucleic acid) or fragmentsthereof, oligonucleotides of any desired base sequences, antigens,antibodies or epitopes, or enzymes, proteins or polypeptide chains whichare a part of the proteins and have the functions, to porous carrierparticles using a binding material.

(8) A method for fabrication of a reactive probe chip, characterized inthat the one or more loaded porous carrier fine particles fabricatedaccording to (5) or (6) above are arrayed and bound or immobilized in atleast one of a plurality of microcompartments provided on a basematerial while maintaining the reactivity of the inner surfaces of theporous carrier particle pores.

According to one aspect of the invention, oligonucleotides having anydesired base sequence or proteins having any desired structure may besynthesized on porous carrier particles with a surface of the bindingability, such as porous glass, silica gel or ion-exchange resin, by asolid phase method. Alternatively, the reactive substances or probessuch as DNA, RNA or PNA (peptide nucleic acid) or fragments thereof,oligonucleotides of any desired base sequences, antigens, antibodies orepitopes, or enzymes, proteins or their fragments staining theirfunctions, are bound to the porous carrier particles using some sort ofbinding material. Thus, the loaded porous carrier particle probes areproduced.

The produced particulate probes may be used alone or in combination, andbound or immobilized utilizing a dispenser or printing method, on atleast one of a plurality of microcompartments provided on a basematerial. The base material is selected from an inorganic base materialsuch as a slide glass or silicon wafer or an organic base material suchas a polyester film or polyethylene film. The probes are bound on thebase material while maintaining the reactivity of the inner surfaces ofthe porous carrier particle pores.

For the binding and immobilization of the porous carrier particles in anorderly fashion on the base material according to the invention, onlythe outer surfaces of the carrier particles are used for immobilization,and a protective measure such as impregnation with water is utilized inorder to accomplish immobilization without incurring damage to the innerpore surfaces by an adhesive component used for the immobilization.

In accordance with another aspect of the invention, the aforementionedproblems can be solved by arraying and binding reactive substances orprobes on tile-like carriers, and then arraying and immobilizing eachtile-like carrier on a base material.

Another aspect of the present invention solves the aforementionedproblems by way of the following means.

(1) A reactive probe chip characterized in that tile-like carriersloaded with reactive substances are arrayed and immobilized on a basematerial.

(2) A reactive probe chip according to (1) above, wherein the tile-likecarriers loaded with the reactive substances are of a material having areactive surface, and then the base material immobilizing the carriersincludes a thin inorganic or organic plate.

(3) A method for fabrication of a reactive probe chip, characterized inthat enzymes, antigens, DNA fragments, antibodies, epitopes or proteinsare arrayed and immobilized on tile-like carriers, and each of theloaded carriers is immobilized in an orderly fashion in separatecompartments on the base material.

(4) A method for fabrication of a reactive probe chip according to (3)above, wherein after synthesizing oligonucleotides with desired basesequences on tile-like carriers, each carrier is immobilized in anorderly fashion into separate compartments on the base material.

(5) A method for fabrication of a reactive probe chip according to (3)or (4) above, wherein the tile-like carriers are each a plate with asquare shape having a size of from 50 μm to 5 mm on each side, or ahexagonal or circular shape, and they are mechanically attached andimmobilized on the base material.

In still another aspect of the present invention, an excellent compositesubstrate that can exhibit a plurality of different functions as asingle substrate, is provided.

The aspect of the present invention has been completed by focusingattention on applicability not found in the prior art and by providing acomposite substrate wherein either a plurality of porous regions arearrayed and comparted by non-porous regions, or a plurality ofnon-porous regions are arrayed and comparted by porous regions on atleast a section of the surface of a porous solid conventionally used inthe form of particles. The composite substrate provides reaction siteswith a plurality of different functions on the same substrate, or tothermally or electrically insulate specific regions of the substrate.

Still another aspect of the present invention solves the aforementionedproblems by way of the following means.

(1) A composite substrate characterized in that on at least a section ofthe surface thereof, a plurality of porous regions are arrayed on asubstrate material and comparted by non-porous regions, or a pluralityof non-porous regions are arrayed and comparted by porous regions.

(2) A composite substrate according to (1) above, wherein a compositesubstrate comprising both porous regions and non-porous regions has asurface flattened by, for example, a polishing process.

(3) A method for fabrication of a composite substrate, wherein thecomposite substrate according to (1) above is produced by situating aseparately formed porous solid at predetermined regions on a non-poroussubstrate.

(4) A method for fabrication of a composite substrate according to (3)above, characterized in that the formation of the composite substrate isaccomplished by situating a plurality of porous solid precursors atpredetermined regions on a non-porous substrate and producing pores inthe porous solid precursors placed on the substrate.

(5) A method for fabrication of a composite substrate according to (3)above, wherein formation of the plurality of porous regions isaccomplished by producing pores in a plurality of predetermined poroussolid precursor regions on a substrate the entire surface of which is aporous solid precursor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration showing a process for fabrication of areactive probe chip using a carrier particle immobilizing apparatusaccording to Example 1.

FIG. 2 is an illustration showing a process for fabrication of areactive probe chip using a carrier particle immobilizing pin accordingto Example 2.

FIG. 3 is an illustration showing a process for fabrication of areactive probe chip using a carrier particle immobilizing pin accordingto Example 3.

FIG. 4 is an illustration showing a process for fabrication of areactive probe chip by screen printing according to Example 4.

FIG. 5 is an illustration of a fabrication process for a reactive probechip according to the invention.

FIG. 6 is an illustration of a fabrication process for an aluminasubstrate having a plurality of porous alumina bead regions on thesurface.

FIG. 7 is an illustration of a fabrication process for a glass platehaving a plurality of porous glass regions on the surface.

FIG. 8 is an illustration of a process for fabrication of a glass platehaving a plurality of porous glass regions by coating of a porous glassprecursor on the surface.

FIG. 9 is an illustration of a process for fabrication of a quartz platehaving a plurality of porous glass regions by a split-phase method withcoating of borosilicate glass particles on the surface.

DETAILED DESCRIPTION OF THE INVENTION

It is a characteristic of one aspect of the present invention that theoligonucleotides constituting the DNA probes are bound on the inner poresurfaces of the porous carrier particles, so that the porous carrierparticles act as “loaded porous carrier particulate probes” as they are.

Another aspect of the present invention is directed to a reactive probechip characterized in that a plurality of particulate or tile-likecarriers loaded with reactive probes are immobilized in separatecompartments on the surface of a base material. Since a plurality ofreactions can be conducted simultaneously, many types of reactive probesare used. The compartments for placing the tile-like carriers arepreferably arranged in such a manner that the plurality of particulateor tile-like carriers loaded with the reactive probes can be arrayed bya mechanical or automatic procedure. The compartments are preferablyformed in well-ordered array.

It further relates to a method for fabrication of a reactive probe chip,characterized in that after synthesizing oligonucleotides having desiredbase sequences on tile-like carriers, each tile-like carrier isimmobilized in separate compartments on a base material. In this case,it is necessary to use means that can rapidly and sequentially convenethe small tile-like carriers loaded with the oligonucleotides fromcontacting the surface in the compartments on the base material.

A composite substrate of still another aspect of the invention has aplurality of porous regions on at least a section of its surface, whichregions are surrounded and comparted by non-porous regions.

Alternatively, the composite substrate has a plurality of non-porousregions that are surrounded and comparted by porous regions.

In the former case, for example, a specific catalytic reaction or enzymereaction can take place only in the compartmented regions. Thus, if asample is exposed to a substrate having a plurality of such regionscomparted isolatedly each other, the sample will be allowed tosimultaneous testing of the presence of reactivity for the plurality ofreactions.

In the latter case, for example, a semiconductor fabrication process maybe employed to partition a plurality of wirings (non-porous regions) byinsulative porous regions and to form a highly insulated electricalwiring on the substrate.

The reactive substances or probes carried on the porous particles may beany of those which react with detection targets, for example,oligonucleotides, enzymes, antigens, antibodies, epitopes or proteins. Amethod wherein the reactive probes immobilized on each of the carriersare arrayed and set in the compartments on the base material surface canstably and flexibly produce the chips.

Two methods exist for loading the reactive substances on the inner poresurfaces of the porous carrier particles. One method is thatoligonucleotides having desired base sequences or proteins with desiredstructures are synthesized on the porous carrier particles by asolid-phase method, to prepare the reactive substances in situ.

The other method is as follows. A purified extract from animal or plantcells or a synthesized reactive substance is bound by some method, forexample, in the case of porous glass, reacting amino silane with theporous glass surface to bind the amino groups. Using glutaraldehyde,various enzymes can be bond to the formed amino groups.

The reaction between the detection target and the reactive substancesoccurs in the pores of the porous carrier particles, and since thedetection target must be incorporated in the pores of the porous carrierparticles, the pores of the porous carrier particles must be largeenough that the detection target can be moved into by the incorporateddiffusion. The pore size usually ranges from 10 nm to 1 μm, andpreferably 50 nm to 200 nm.

Reaction in the pores of the porous carrier particles can preventside-reaction with contaminants, and the larger reactive surface areaallows more accurate detection.

According to one aspect of the invention, highly stable, reactivesubstance-loaded porous carrier particles or tile-like plates can bestored because the reactive substance synthesized on the carrier is usedas it is, or because the substances immobilized on the carriers in usedin the detection.

While there are no particular restrictions on the method of arraying andimmobilizing the reactive substance on the base material, a protectivesolution, such as a single amount of water may be contained in thecarrier particles to protect the inner surface of the pores and then aninorganic base material such as silica sol is added to the carrierparticles to make a slurry, which is arrayed on a support by use of adispenser. This method is useful to fabricate a small number of reactiveprobe chips, but in order to mass-produce the reactive probe chips, theslurry may be used in the form of printing ink to form the array patternby a multi-color printing method.

When using such methods, it will be difficult to form a slurry or ink ifthe size of the “loaded porous carrier particles” is very large, andtherefore the porous carrier particle size preferably ranges from 1 μmto 100 μm, and more preferably from 3 μm to 20 μm. This is because alarge particle size is preferred from the standpoint of handleabilityduring the process of loading the reactive substance, but a smallparticle size is preferred when the porous carrier particles areimmobilized after the reactive substances have been loaded;nevertheless, large grains may be used so long as they can be arrayed.

By using these methods it is possible to achieve stable and flexibleproduction. The base material may be of a material that is stable and isnot deteriorated upon using it in the detection system, but it must havesurface properties suitable for immobilizing the porous carrierparticles. Glass plates such as quartz glass, borosilicate glass and thelike, or inorganic base materials such as silicon wafers are preferred.

Modification of the method of binding the porous carrier particles willenable to use of an organic base material such as a polyester film orpolyethylene film, and in some cases even paper materials can be used.The base material surface may be appropriately treated for the purposeof adjusting the affinity with the carrier binding material.

The porous carrier particles must be of a material that can carry, asthe reactive substances, any proteins having the desired structures orthe oligonucleotides having the desired base sequences. The porousmaterials having a binding ability, such as porous glass, silica gel,ion-exchange resin and the like are preferred, among which porous glassis most preferred because its surface reactivity associated with poresizes can be controlled.

The surface of the porous carrier particle is preferably subjected toappropriate surface treatment in order to adjust its affinity with thereactive substance or reactive probe.

There are no particular restrictions on the shape of the base material,and for example, it may be thin plate such as a film or sheet, or in acubical, rod-like, cord-like or spherical shape.

When a thin plate is used, there are no particular restrictions on thethickness or size of the base material, and the thickness of the basematerial may be easily determined in consideration of the form stabilityrequired for the base material. The size of the base material may beeasily determined on consideration of the number of microcompartments tobe formed in the base material surface.

The microcompartments on the base material surface according to theinvention are imaginary compartments, which are defined by imaginarilyformed partitions and not materially separated.

The “reactivity” of the “reactive substances or reactive probes”according to the invention refers to not only a change in their chemicalstructure by ionic bonding or covalent bonding through a chemicalreaction, but also the property capable of forming binding states withother substances due to Van der Waals forces, hydrogen bonding,coordination bonding, chemical adsorption, physical adsorption or thelike.

Such reactive substances, which are alternatively referred to asreactive probes, include proteins with any desired structures andoligonucleotides with any desired base sequences, and naturally thereare no restrictions on these.

There are no particular restrictions on the degree of integration of themicrocompartments, i.e., the compartments for the reactive substances,in a reactive probe chip according to the invention. Since the degree ofintegration required or convenient will differ depending on the use ofthe reactive probe chip, the degree of integration may be appropriatelychanged to conform to the use.

As an example, there may be 100 or more micro-compartments per cm² ofthe reactive probe chip surface, and if the base material and reactivesubstances are appropriately selected, about 10,000 microcompartmentsper cm² of surface can be formed.

Since the reactive probe chip of the invention has the reactivesubstances loaded on porous carrier particles, tiles or the like, thesubstance are not easily penetrated and released on the base material.Also, since the size of porous carrier particles is small, it ispossible to immobilize a solution containing the particles at highdensity in the compartments on the base material.

The reactive substances carried on the porous carrier solid may be thesame types of substances or different types of substances, depending onthe use of the reactive probe chip. From the viewpoint of workingefficiency, it is preferred to load a plurality of reactive substancesat one time, and it is more preferred to load all of the reactivesubstances at one time.

The reactive substance-loaded porous carrier solid may be separatelyprepared and stored, or if necessary, they may be immobilized on thebase material in the necessary combinations. Especially, it is practicalin the case of oligonucleotide-synthesized porous carrier particles ortiles, because a usual synthesis process can be used.

The size and shape of the carriers may be selected as desired, butconsidering immobilization of the carriers loaded with a plurality ofdifferent reactive probes onto the substrate, when tile-like carrier isused, it is preferably a plate with a square shape having a size of from50 μm to 5 mm on each side, or a hexagonal or circular shape, andsquares of 100 μm to 1 mm are particularly preferred. The thickness willdepend on the size, but it is preferably in the range of 100-200 μm. Theimmobilization of the tile-like carriers on the base material may bemechanically attached and immobilized on the substrate using an adhesivethat does not affect the reaction.

For example, an acrylic resin may be used as an adhesive forimmobilization of the tile-like carriers onto the substrate.

The apparatus used for immobilization of the tile-like carriers onto thesubstrate may be one used for processing and conveyance of micromembersused in the production of semiconductor devices.

In case the tile-like carrier is used, a process for fabrication of areaction chip according to the invention will now be explained withreference to the attached drawings.

FIG. 5 is an illustration of the fabrication process, and in step (a),an adhesive 2 is coated on the area in which the reactive probes are tobe set on the glass slide 1 serving as the base material for thereaction chip.

In step (b), an array dispenser 4 with a suction chuck 5 at the lowerend is moved to Region A which contains base materials bearing aplurality of the same reactive probe-loaded tiles 3 manufactured inadvance, and a reactive probe-loaded tile 3 is suctioned with thesuction chuck 5, and then in step (c), the array dispenser 4 is movedover the glass slide 1, disengaging the suction for ejection at theprescribed location, and immobilizing the reactive probe-loaded tile 3it has carried onto the prescribed location.

Then, in step (d), a tile 3 is conveyed by the array dispenser 4 fromRegion B which contains substrates bearing a different plurality ofreactive probe-loaded tiles 3 and is immobilized at the subsequentprescribed location, and this operation is repeated in order to arrangedifferent types of reactive probe-loaded tiles 3.

This yields, in step (e), a reaction chip 6 with different types ofreactive probe-loaded tiles 3 orderly arranged at prescribed locations.

The porous solid for producing a composite substrate of the stillanother aspect of the invention is preferably porous glass or porousceramic that enables to easily control the pore size and to easilyattach functional groups thereto. Split-phase porous glass with a highdensity of surface hydroxyl groups, which readily undergoes chemicalmodification, is especially suitable for use as a reaction site. Thesample can also be rendered homogeneous by flattening the surface by aprocess such as polishing.

The split-phase porous glass has properties that allow easy control ofthe pore size by appropriately selecting the heat treatment time andtemperature, and borosilicate glass is preferred as the parent glasscomposition.

Porous glass compositions include Na₂O—B₂O₃—SiO₂ based glass with SiO₂in a range of 55-80 wt % or SiO₂ in a range of 35-55 wt %, as well asSiO₂—B₂O₃—CaO—Al₂O₃ based glass, SiO₂—P₂O₅—Na₂O based glass,SiO₂—B₂O₃—CaO—MgO—Al₂O₃—TiO₂ based glass, SiO₂—B₂O₃—Na₂O—GeO₂ basedglass, SiO₂—ZrO₂ based glass and GeO₂—ZrO₂—ThO₂ based glass.

As porous ceramics there may be mentioned alumina, magnesia and thelike.

Several differing methods may be used as fabrication methods for thecomposite substrate of the invention.

One is a method whereby a separately formed porous solid is situated onprescribed regions of a non-porous substrate. The porous solid used heremay be particulate, or crushed or cut fragments of a porous solid plate.The porous solid may also be given a necessary function, for example, byloading a reactive probe or reactive substance on the porous solid,prior to being arrayed on the substrate.

For arraying on the substrate, it is preferred to use an adhesive thatdoes not affect the function of the reactive probes or the porous solid,such as water glass. Pits may also be preformed in the base material andthe porous solid embedded therein, and then the entire substratesubjected to polishing to create a flat composite substrate.

Another method is one whereby a plurality of porous solid precursors(substances) are situated on prescribed regions of a non-poroussubstrate and pores are produced therein on the base material. Themethod of situating the precursors may be a method of attachment ofsolids with an adhesive or the like, or a method of dropping a liquid orslurry and heat treating it to form a precursor (for example a precursorsubstance layer) attached to the substrate.

In this case as well, pits may be preformed at prescribed locations inthe substrate and the porous solid precursors embedded therein, andpolishing carried out after production of the pores to obtain a flatcomposite substrate.

Another possible method involves preparing a substrate composed of aporous solid precursor, or forming a porous solid precursor layer orthin-film over the entirety of a non-porous substrate, and producingpores in prescribed regions thereof.

The method of forming the precursor on the non-porous substrate may be amethod of attachment of a plate with an adhesive, uniform coating of asolution or slurry by pin coating or the like, or attachment on thesubstrate by heat treatment or the like. For production of the pores, aresist may be used to cover the other sections in order to produce poresonly in the prescribed locations. In this case, the surface of thesubstrate is relatively flat prior to pore production, but if necessaryit may be further flattened by polishing.

By preparing a substrate which is porous over the entirety andeliminating the pores in prescribed regions of the surface by sealingtreatment, with the prescribed regions selected at locations that formthe borders for the porous sections so that they are separated by aplurality of compartments, it is possible to form a surface with aplurality of porous regions compartmented by non-porous formed regions.

The sealing method used to accomplish this may be a method ofirradiation of the prescribed regions with a high energy beam such as alaser beam, or a method of chemical treatment after coating the regionsto be left porous with a resist. Laser processing is preferred becauseonly the regions exposed to the laser are melted, and there is lesseffect on the other locations. Laser processing can also easily form aplurality of compartmented regions by simply shifting the laser atspacings on the substrate to draw horizontal and vertical lines.

The present invention will be illustrated in detail by way of examples.It is to be understood, however, that the invention is in no way limitedby these examples.

EXAMPLE 1

Proteins with different structures were synthesized on particles of asurface-aminated ion-exchange resin powder having a mean particle sizeof 10 μm and an average pore size of 10 nm.

The protein-loaded porous ion-exchange resin powder was dispersed inpurified water and silica sol was added to prepare a slurry. The slurrywas then loaded in each of 1 mm-square compartments of the surface of aglass plate 4 composed of a borosilicate glass slide (approximately 15cm×2 cm), using the ultrathin capillary of the carrier particle bindingapparatus (dispenser) 3 shown in FIG. 1. A reactive probe chip capableof carrying out reactions with 750 different proteins was thusfabricated.

EXAMPLE 2

Different oligonucleotides were synthesized by an established method onaminosilylated silica gel particles 5 for liquid chromatography having adiameter of 3 nm and a pore size of 10 nm.

A slurry prepared by adding an aqueous polyvinyl alcohol solution tothis oligonucleotide-immobilized silica gel was held onto the carrierparticle-immobilizing pin 7 shown in FIG. 2, and then immobilized at apitch 0.5 mm on the surface of a ribbon-shaped silica gel coatedpolyester film 8 having a size of approximately 0.5 cm×20 cm, to obtaina reactive probe chip according to the invention.

EXAMPLE 3

Porous glass powder with a pore size of 50 nm and a diameter of 5 μmwith a γ-aminopropylsilylated surface was used to synthesize differentoligonucleotides by an established method.

A slurry prepared by adding an acryl polymer to theoligonucleotide-loaded porous glass powder was held on the carrierparticle-immobilizing pin 7 shown in FIG. 3, and then arrayed andimmobilized at a pitch of 0.5 mm on the surface of an oxide film-coatedsilicon chip 11 having a size of approximately 1 cm×1 cm, to obtain areactive probe chip according to the invention.

EXAMPLE 4

Porous glass powder with a pore size of 100 nm and a diameter of 5 μmwith a γ-aminopropylsilylated surface was used to synthesize differentoligonucleotides by an established method.

An acryl polymer was added to each of the porous glass powders loadedwith different oligonucleotides, to prepare pastes containing porousglass powders loaded with the different oligonucleotides. The pastes 13were immobilized at a pitch of 0.5 mm on the surface of a slide glass 14(approximately 1 cm×1 cm), which had been subjected to a surfaceblasting treatment to make the surface delustered. The immobilizationwas carried out by using a multicolor screen printing technique, andthis printing was repeated (1-n) times to obtain a reactive probe chip15 according to the invention.

EXAMPLE 5

An enzyme with a certain specific reactivity was immobilized on a 1mm-square, 10 μm-thick surface aminated “cover glass” usingglutaraldehyde. A “cover glass” loaded with different enzymes was alsoprepared in the same manner, and these were consecutively arranged on anacrylic adhesive-coated glass slide having a length of 75 mm, a width of25 mm and a thickness of 1.5 mm, using an aligning apparatus which was amodified semiconductor wire bonder. These were loaded into each ofseveral 1 mm-square compartments. A reactive probe chip capable ofcarrying out 100 different antigen enzyme reactions was thus fabricated.

EXAMPLE 6

Different oligonucleotides were synthesized by an established method onsurface-aminated porous glass with a size of 0.5 mm square and athickness of 10 μm. The oligonucleotide-immobilized porous glass wasorderly arranged on an acrylic adhesive-coated glass slide withdimensions of 75 mm length, 25 mm width and 1.5 mm thickness. A1000-type complementary DNA detection chip was fabricated.

EXAMPLE 7

Different oligonucleotides were synthesized by an established method onsurface-aminated porous glass with a size of 0.5 mm square and athickness of 10 μm. The oligonucleotide-immobilized porous glass wasorderly arranged on the surface of an epoxy adhesive-coated polyesterfilm (approximately 3 cm×20 cm, 0.3 mm thickness) at a 0.5 mm pitch, toobtain a reactive probe chip according to the invention.

EXAMPLE 8

As shown in FIG. 6, an alumina substrate 1 with dimensions of 100 mmlength×100 mm width×1 mm thickness was coated with silica sol to form asilica sol layer 2, and then porous alumina beads 3 with a mean particlesize of 50 μm were dispersed thereover and dried for immobilization.

The porous alumina beads 3 had a void volume of 40% and a surface areato weight ratio of 250 m²/g, and the individual particles can carryagents with different functions.

EXAMPLE 9

As shown in FIG. 7, a plurality of pits 5 with dimensions of 3 mmlength×3 mm width and 0.1 mm depth were formed by wet etching onprescribed locations of the surface of a glass plate 4 with dimensionsof 75 mm length, 25 mm width×1.5 mm thickness. Polyacrylamide 6 wasplaced in the pits 5.

A 0.2 mm-thick porous glass plate 7 was cut into sizes insertable in thepits 5, and the porous glass fragments 8 were placed on thepolyacrylamide 6 in the pits 5, and attached therein. This caused aportion of the polyacrylamide 6 to spill over the edges of the pits 5,or else the tops of the porous glass fragments 8 protruded above thesurface of the glass plate 4. The attached substrate is subjected toflattening treatment if necessary.

This method yielded a glass plate 4 having polyacrylamide 6 in aplurality of pits 5, with porous glass fragments 8 immobilized by thepolyacrylamide 6.

By dropping a function-providing reagent onto the porous glass fragment8 sections using a micropipette, it is possible to manufacture afunctional glass plate.

If the properties of the functional substances carried on the porousglass fragments 8 are such that the functional substances are affectedby the polyacrylamide, the polyacrylamide may be changed to anotheradhesive polymer.

EXAMPLE 10

As shown in FIG. 8, a plurality of pits 5 with dimensions of 3 mmlength×3 mm width and 0.2 mm depth were formed by wet etching onprescribed locations of the surface of a glass plate 4 with dimensionsof 75 mm length, 25 mm width×1.5 mm thickness.

After adding 2 ml of water, 4 ml of formamide and 0.2 ml of 12 Nhydrochloric acid to a solution of 2 ml tetraethyl silicate in 2 ml ofethyl alcohol in a separate stirring tank, for hydrolysis of thetetraethyl silicate, it was aged for 5 hours at room temperature andthen a highly viscous gel 10 of a porous glass precursor was formed.

This was spin coated onto the surface of a glass plate 4 (to 0.3 mmthickness), aged and dried, to form a porous glass precursor layer 11.Next, the porous glass precursor layer 11 sections were polished toremove the sections raised from the surface of the glass plate 4, togive a glass plate having a porous glass precursor 12 only on thesections of the pits 5.

Finally, the porous glass precursor 12 was treated to convert it toporous glass 13, to give a composite glass plate 14 having a pluralityof porous glass regions in non-porous glass regions.

EXAMPLE 11

As shown in FIG. 9, a plurality of pits 5 with dimensions of 3 mmlength×3 mm width and 0.15 mm depth were formed by cutting on prescribedlocations of the surface of a quartz plate 15 with dimensions of 75 mmlength, 30 mm width×1.5 mm thickness.

Borosilicate glass particles with a mean particle size of 10 μm and anoil (paraffin wax) were placed in an stirring tank 16 and stirred toform a coating solution, which was then coated onto the surface of thequartz plate 15 by screen printing to form a coating layer 17. Thecoating coverage was 400 g/M².

This was dried and heat treated to remove the oil portion, subsequentlyheated at 850° C. for 30 minutes to fused together the borosilicateglass particles, and then heat treated at 600° C. for 15 hours forsplit-phase treatment. The borosilicate glass sections 18 were thenpolished to remove the sections raised above the surface of the glassplate 10, to give a quartz glass plate 19 having borosilicate glass 18only in the sections of the pits 5.

This quartz glass plate 19 was acid treated for 0.5 hour using sulfuricacid (1 N concentration) at approximately 90° C. to produce pores, thusgiving a composite quartz glass plate 21 having a plurality of porousborosilicate glass regions 20.

According to the invention, it is possible to easily and cheaply providea reactive probe chip having reactive probes such as DNA fragmentsintegrated on its surface, without the need for special equipment suchas photolithography equipment. Also, selection of the base material andmodifications to the method of loading the reactive probes can provide achip with a higher degree of integration than existing DNA chips.

By preparing a carrier loaded with different reactive probes, it ispossible to more conveniently supply chips loaded with the necessarycombinations of DNA probes for necessary occasions, while also allowingconstruction of a reactive probe chip with different types of reactiveprobes immobilized and thereby providing a DNA chip of lower cost andhigher stability that can be helpful in the clinic for detection ofindividual DNA polymorphisms.

According to another aspect of the invention there is provided acomposite substrate wherein a plurality of porous regions are orderlyarranged as compartments on at least a section of the surface thereof,and therefore a plurality of different functions or performances can beexhibited on the same substrate, thus offering applicability not foundin the prior art.

For fabrication of the composite substrate, porous glass fragments areimmobilized onto the substrate at a prescribed spacing via an adhesivelayer, or anchorable sites such as pits are formed in the substrate at aprescribed spacing and the porous glass fragments are immobilizedtherein, as a simple means of orderly arranging a plurality of porousregions in compartments.

1. A reactive probe chip for detecting target functional molecules,comprising: one or a plurality of first carrier probes, wherein saidfirst carrier probe is a porous carrier in the form of a particle,having immobilized within the pores thereof a first reactive substancecapable of bonding a first target molecule; one or a plurality of secondcarrier probes, wherein said second carrier probe is a porous carrier inthe form of a particle, having immobilized within the pores thereof asecond reactive substance capable of bonding a second target molecule;and a substrate material, wherein said one or a plurality of firstcarrier probes and said one or a plurality of second carrier probes areimmobilized on a surface of said substrate material.